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Objective: Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice., Methods: Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology., Results: Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (-49%; p=0.01) and mouse lymphocytes (-41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (-86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001)., Conclusions: These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3(+) autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4(S27N) had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4(Q72L) showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4(S204Q) showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab4(1-121), showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab4(1-121) increased the formation of LC3(+) autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab4(1-121), HRES-1/Rab4(Q72L) and HRES-1/Rab4(S204Q) promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4(S27N) mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4(Q72L) upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3(+) autophagosomes and the preservation of mitochondria.

TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.

Activation, proliferation, or programmed cell death of T lymphocytes are dependent on controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (Delta Psi(m)) also plays a decisive role in cell survival by controlling activity of redox-sensitive caspases. T lymphocytes of patients with systemic lupus erythematosus (SLE) exhibit mitochondrial hyperpolarization, increased ROI production, diminished intracellular glutathione levels, cytoplasmic alkalinization, and ATP depletion that mediate enhanced spontaneous and diminished activation-induced apoptosis and sensitize lupus T cells to necrosis. These redox and metabolic checkpoints represent novel targets for pharmacological intervention in SLE.

Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within the immune system. Activation, proliferation, or death of cells of the immune system are dependent on controlled reactive oxygen intermediate (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (Delta(Psi)m) reflects the energy stored in the electrochemical gradient across the inner mitochondrial membrane, which in turn is used by F0F1-ATPase to convert adenosine 5'-diphosphate to ATP during oxidative phosphorylation. Mitochondrial hyperpolarization and transient ATP depletion represent early and reversible steps in T-cell activation and apoptosis. By contrast, T lymphocytes of patients with SLE exhibit elevated Delta(Psi)m, that is, persistent mitochondrial hyperpolarization, cytoplasmic alkalinization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP. Oxidative stress affects signaling through the T-cell receptor as well as the activity of redox-sensitive caspases. ATP depletion may be responsible for diminished activation-induced apoptosis and sensitize lupus T cells to necrosis. Mitochondrial dysfunction is identified as a key mechanism in the pathogenesis of SLE.

Objective: Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (deltapsim) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE., Methods: We assessed the deltapsim with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin-luciferase assay., Results: Both deltapsim and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased deltapsim and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and deltapsim elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the deltapsim elevation in lupus. CD3:CD28 costimulation led to transient elevation of the deltapsim, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1-ATPase, had similar effects., Conclusion: T cell activation and apoptosis are mediated by deltapsim elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE.

Redox mechanims play important roles in replication of human immunodeficiency virus type 1 (HIV-1) and cellular susceptibility to apoptosis signals. Viral replication and accelerated turnover of CD4+ T cells occur throughout a prolonged asymptomatic phase in patients infected by HIV-1. Disease development is associated with steady loss of CD4+ T cells by apoptosis, increased rate of opportunistic infections and lymphoproliferative diseases, disruption of energy metabolism, and generalized wasting. Such pathological states are preceded by: (i) depletion of intracellular antioxidants, glutathione (GSH) and thioredoxin (TRX), (ii) increased reactive oxygen species (ROS) production, and (iii) changes in mitochondrial transmembrane potential (deltapsi(m)). Disruption of deltapsi(m) appears to be the point of no return in the effector phase of apoptosis. Viral proteins Tat, Nef, Vpr, protease, and gp120, have been implicated in initiation and/or intensification of oxidative stress and disruption of deltapsi(m). Redox-sensitive transcription factors, NF-kappaB, AP-1, and p53, support expression of viral genes and proinflammatory lymphokines. ROS regulate apoptosis signaling through Fas, tumor necrosis factor (TNF), and related cell death receptors, as well as the T-cell receptor. Oxidative stress in HIV-infected donors is accompanied by increased glucose utilization both on the cellular and organismal levels. Generation of GSH and TRX from their corresponding oxidized forms is dependent on NADPH provided through the pentose phosphate pathway of glucose metabolism. This article seeks to delineate the genetic and metabolic bases of HIV-induced oxidative stress. Such understanding should lead to development of effective antioxidant therapies in HIV disease.

Stimulation of the CD95/Fas/Apo-1 receptor leads to apoptosis through activation of the caspase family of cysteine proteases and disruption of the mitochondrial transmembrane potential (Deltapsim). We show that, in Jurkat human T cells and peripheral blood lymphocytes, Fas-induced apoptosis is preceded by 1) an increase in reactive oxygen intermediates (ROI) and 2) an elevation of Deltapsim. These events are followed by externalization of phosphatidylserine (PS), disruption of Deltapsim, and cell death. The caspase inhibitor peptides, DEVD-CHO, Z-VAD.fmk, and Boc-Asp.fmk, blocked Fas-induced PS externalization, disruption of Deltapsim, and cell death, suggesting that these events are sequelae of caspase activation. By contrast, in the presence of caspase inhibitors, ROI levels and Deltapsim of Fas-stimulated cells remained elevated. Because ROI levels and Deltapsim are regulated by the supply of reducing equivalents from the pentose phosphate pathway (PPP), we studied the impact of transaldolase (TAL), a key enzyme of the PPP, on Fas signaling. Overexpression of TAL accelerated Fas-induced mitochondrial ROI production, Deltapsim elevation, activation of caspase-8 and caspase-3, proteolysis of poly(A)DP-ribose polymerase, and PS externalization. Additionally, suppression of TAL diminished these activities. Therefore, by controlling the balance between mitochondrial ROI production and metabolic supply of reducing equivalents through the PPP, TAL regulates susceptibility to Fas-induced apoptosis. Early increases in ROI levels and Deltapsim as well as the dominant effect of TAL expression on activation of caspase-8/Fas-associated death domain-like IL-1beta-converting enzyme, the most upstream member of the caspase cascade, suggest a pivotal role for redox signaling at the initiation of Fas-mediated apoptosis.

The nephrotoxic cysteine S-conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) is metabolized by kidney homogenates and subcellular fractions to pyruvate and a reactive thiol, which is cytotoxic and partially decomposes to yield hydrogen sulfide and thiosulfate. Although hydrogen sulfide is a potent mitochondrial poison, the mitochondrial toxicity of CTFC is not attributable to hydrogen sulfide formation, as shown by different sites of inhibition of mitochondrial respiration by CTFC and hydrogen sulfide. The efficient mitochondrial oxidation of hydrogen sulfide apparently serves to protect mitochondria against the toxic effects of hydrogen sulfide generated from CTFC.